Intracellular pathogens are responsible for an extensive amount of morbidity and mortality world-wide. Our current understanding of host reponse to intracellular pathogens stems mainly from extensive analysis of murine cell-mediated immunity to the facultative intracellular bacterial pathogen Listeria monocytogenes. Athough the immune response to L. monocytogenes has received enormous attention surprising little research has been devoted to understanding the cell biology of intracellular growth or to bacteria determinants of pathogenicity. The overall goal of the proposed research is to define in molecular terms listerial determinants required for intracellular growth. One likely determinant of L. monocytogenes pathogenesis is the elaboration of a sulfhydryl-activated hemolysin. In preliminary studies, conjugative transposons were used to isolate non- hemolytic mutants. The mutants fail to grow in the mouse macrophage like cell line J774. In the proposed study, the mutants will be evaluated with respect to growth in vivo as well as intracellularly, in vitro. Preliminary data support that L. monocytogenes grows freely in the eucaryotic cell cytoplasm. Electron microscopy of thin-sectioned infected cells will be used to evaluated the role of hemolysin for intracellular localization. As an initial step in structure-function analysis of the hemolysin, the hemolysin gene will be cloned from L. monocytogenes cosmid DNA libraries in E. coli. The primary amino acid sequnce will be deduced from the nucleotide sequence and compared with other pore-forming proteins. The cloned gene will also be used as a hybridization probe to examine its conservation among Listeria species. The hemolysin gene will be cloned into a streptococcal shuttle vector, transformed into a streptococcal strain and conjugated to a non-hemolytic strain of L. monocytogenes. This methodology will facilitate future work in which in vitro constructed hemolysin mutations can be studied in their normal background. Genes other than hemolysin are likely to contribute to listerial infectivity. An attempt will be made to isolate tansposon insertions in genes other than hemolysin which are required for intracellular multiplication. Mutants will be selected inside tissue culture cells in the presence of ampicillin, an antibiotic that kills only growing bacteria.